The present invention relates to recombinant enzymes, particularly, novel recombinant alcohol/aldehyde dehydrogenases (hereinafter referred to as AADH or AADHs) having alcohol and aldehyde dehydrogenase activity. The present invention also relates to novel recombinant DNA molecules encoding AADHs, recombinant expression vectors containing said DNAs, and recombinant organisms containing said recombinant DNA molecules and/or said recombinant expression vectors. Furthermore, the present invention relates to a process for producing recombinant AADHs and a process for producing aldehydes, carboxylic acids and ketones, especially, 2-keto-L-gulonic acid (herein after referred to as 2KGA) by utilizing said recombinant enzymes, and a process for producing aldehydes, carboxylic acids and ketones, especially, 2KGA by utilizing said recombinant organisms.
2-KGA is an important intermediate for the production of L-ascorbic acid (vitamin C). For example, 2KGA can be converted into ascorbic acid according to the well-known Reichstein method Numerous microorganisms are known to produce 2KGA from D-sorbitol or L-sorbose. Japanese Patent Publication No. 51-40154 (1976) discloses the production of 2KGA from D-sorbitol by microorganisms of the genus Acetobacter, Bacterium or Pseudomonas. According to Acta Microbiologica Sinica 21(2), 185-191 (1981), 2KGA can be produced from L-sorbose by a mixed culture of microorganisms, especially, Pseudomonas striata and Gluconobacter oxydans. European Patent Publication No. 0221 707 discloses the production of 2KGA from L-sorbose by Pseudogluconobacter saccharoketogenes with and without concomitant bacteria. European Patent Publication No. 0278 447 discloses a process for the production of 2KGA from L-sorbose by a mixed culture, which is composed of strain DSM No. 4025 (Gluconobacter oxydans) and DSM No. 4026 (a Bacillus megaterium strain). European Patent Publication No. 88116156 discloses a process for the production of 2KGA from L-sorbose by Gluconobacter oxydans DSM No. 4025.
From G. oxydans DSM No. 4025, AADH was purified and characterized to catalyze the oxidation of alcohols and aldehydes, and was thus capable of producing the corresponding aldehydes and ketones from alcohols, and carboxylic acids from aldehydes (see European Patent Publication No. 606621). More particularly, the AADH catalyzed the oxidation of L-sorbose to 2KGA via L-sorbosone. The physico-chemical properties of the purified sample of the AADH were as follows:
a) Optimum pH: about 7.0-9.0
b) Optimum temperature: about 20xc2x0 C.-40xc2x0 C.
c) Molecular weight: 135,000+/xe2x88x925,000 dalton (Consisting of two subunits in any combination of such xcex1-subunit and xcex2-subunit, each having a molecular weight of 64,500+/xe2x88x922,000 and 62,500+/xe2x88x922,000, respectively)
d) Substrate specificity: active on primary and secondary alcohols and aldehydes including L-sorbose, L-sorbosone, D-sorbitol, D-glucose, D-mannitol, D-fructose, DL-glyceraldehyde, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 2-propanol, 2-butanol, propionaldehyde, PEG1000, PEG2000, PEG4000, PEG6000 and polyvinyl alcohol
e) Prosthetic group: pyrroloquinoline quinone
f) Isoelectric point: about 4.4
Once the genes coding for said AADH have been cloned, they can be used for the construction of a recombinant organism capable of producing a large amount of the recombinant AADH or the various aldehydes, ketones and carboxylic acids, especially, 2KGA. However, there have been no reports so far of the cloning of such genes.
The present invention relates to novel recombinant AADHs having alcohol and aldehyde dehydrogenase activity. Comprised by the present invention are novel recombinant molecules encoding the AADHs; recombinant expression vectors containing said DNAs; recombinant organisms carrying the DNAs and/or recombinant expression vectors; a process for producing the recombinant AADHs; and a process for producing aldehydes, carboxylic acids and ketones, especially, 2KGA utilizing the recombinant AADHs or the recombinant organisms.
More particularly, an aspect of the present invention concerns a recombinant enzyme having alcohol and aldehyde dehydrogenase activity which comprises one or more recombinant polypeptides which contain an amino acid sequence selected from SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and functional derivatives thereof which contain addition, insertion, deletion and/or substitution of one or more amino acid residues, wherein the recombinant polypeptides have said alcohol and aldehyde dehydrogenase activity.
The present invention also provides AADH enzymes which comprise chimeric recombinant polypeptides that are a chimeric combination of at least two of the following amino acid sequences identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and functional derivatives thereof which contain addition, insertion, deletion and/or substitution of one or more amino acid residues, wherein the recombinant polypeptides have said alcohol and aldehyde dehydrogenase activity.
Another aspect of the present invention concerns a recombinant DNA molecule encoding at least one recombinant polypeptide containing an amino acid sequence selected from SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, chimeric combinations of at least two of the following amino acid sequences identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and functional derivatives thereof which contain addition, insertion, deletion and/or substitution of one or more amino acid residues, wherein said recombinant polypeptides have said alcohol and aldehyde dehydrogenase activity.
The recombinant DNA molecules of the present invention contain DNA sequences encoding the polypeptides with alcohol and aldehyde dehydrogenase activity as disclosed, e.g., in the sequence listings herein as well as their complementary strands, or those which include these sequences, DNA sequences which hybridize under standard conditions with such sequences or fragments thereof, and DNA sequences which because of the degeneracy of the genetic code, do not hybridize under standard conditions with such sequences but which code for polypeptides having exactly the same amino acid sequence.
Further aspects of the present invention concern a recombinant expression vector which carries one or more of the recombinant DNA molecules defined above and a recombinant organism which carries the recombinant expression vector defined above and/or carries one or more recombinant DNA molecules on a chromosome.
A further aspect of the present invention concerns a process for producing a recombinant enzyme having an alcohol and aldehyde dehydrogenase activity as defined above, which comprises cultivating a recombinant organism defined above in an appropriate culture medium and recovering said recombinant enzyme.
Another aspect of the present invention concerns a process for producing an aldehyde, ketone or carboxylic acid product from a corresponding substrate which comprises converting said substrate into the product by the use of a recombinant organism as defined above.
Moreover another aspect of the present invention concerns a process for producing 2-keto-L-gulonic acid which comprises the fermentation of a recombinant organism as defined above in an appropriate medium containing L-sorbose and/or D-sorbitol.
Another aspect of the present invention concerns a process for producing an aldehyde, ketone or carboxylic acid product from a corresponding substrate which comprises the incubation of a reaction mixture containing a recombinant enzyme of the present invention.
Furthermore another aspect of the present invention concerns a process for producing 2-keto-L-gulonic acid which comprises the incubation of a reaction mixture containing a recombinant AADH and L-sorbose and/or D-sorbitol.
It is also an object of the present invention to provide an intermediate, i.e., 2-keto-L-gulonic acid, for the production of vitamin C whereby a process for the production of 2-keto-L-gulonic acid as described above is effected and the 2-keto-L-gulonic acid obtained by such process is transformed into vitamin C (L-ascorbic acid) by methods known in the art.